Development Of New Chemotherapeutics For Tuberculosis

National Institute Of Allergy And Infectious Diseases

Investigators

Linked publications & trials

Abstract

Currently this project focuses on five key areas: (1) chemical synthesis of lead molecules and series identified by high-throughput screening against whole Mycobacterium tuberculosis (MTb) under in vivo relevant conditions, (2) clinical candidate development of an oxazolidinone with optimized activity against MTb, (3) identification of environmental microbes that produce anti-tubercular secondary metabolites, (4) unraveling the mechanism of action of hits of interest as well as the mechanisms by which the pathogen adapts to the xenobiotic stress either through modulation of compound uptake, compound metabolism or mutations in the target pathway and in (5) we are exploring the physiological function of important mycobacterial enzymes and microbial biochemistry underlying host pathogenesis. In Project (1) in which we are screening compound libraries obtained from global collaborators including pharmaceutical companies to identify inhibitors of MTb growth under in vivo relevant conditions, performing dose-titration follow-up of hits and synthesizing or purchasing chemically similar compounds. These series are evaluated using secondary screens with a battery of conditions that are thought to be relevant during in vivo growth of MTb. Since September 2020, we have progressed scaffolds of interest that were not flagged as undesirable in our hit triage strategy through formal hit assessment. Our 4-tiered hit prioritization approach bins compounds into major mechanistic classes, in particular highlighting those compounds that hit well-known targets in cell wall synthesis or respiration and excluding generally cytotoxic compounds. Every attempt was made to progress as many chemo-types as possible to increase the likelihood of hitting a diversity of targets. Hit series with multiple members showing activity for the scaffold with low-complexity, acceptable solubility and promising physicochemical properties for profiling are prioritized for follow-up to determine if the desirable balance of potency and ADME (absorption, distribution, metabolism and excretion) properties could be achieved in Lead Optimization. In contrast, series with structural alerts suggesting toxicophores are deprioritized. To rapidly expand the SAR for the prioritized chemotypes, commercially available analogs are purchased and tested in MIC assays. In addition, selected compounds are synthesized to explore preliminary SAR. Kinetic and thermodynamic solubility determinations and microsomal stability assays are also done to further develop the information that will be essential to facilitate go / no-go progression into lead optimization. In project 2, we are working on developing an oxazolidinone with an increased potency against MTb combined with a lower ability to inhibit mitochondrial protein synthesis in order to decrease the toxicities associated with linezolid chemotherapy. We have identified two lead compounds that are less toxic to the host and more potent against the pathogen than linezolid. PK/PD analysis, mouse efficacy and animal toxicity studies suggested that one of these was superior to linezolid with projected human doses greater than 5-fold lower than linezolid. One of these leads progressed to official development status and is entering clinical testing. In project 3, we have identified environmental reservoirs that are rich in mycobacteria that compete with other environmental bacteria for limited nutrients. Specifically, sphagnum peat bogs have been reported to support diverse bacterial and fungal communities including slow-growing mycobacteria closely related to MTb that compete for nutrients under conditions that recapitulate some of the defining characteristics of human granulomas including an acidic pH, hypoxia as well as nutrient limitation. We have the largest global collection of acidobacteria with this library currently being typed based on antitubercular activity. In parallel, we have processed fungal as well as lichen samples as a novel reservoir of potential antibiotic-producing organisms. We have screened extracts from these organisms not only against MTb but also other important bacterial pathogens. To enrich for organisms that produce antibiotics that are selective for MTb, we have screened the microorganisms during co-culture with MTb with subsequent anti-tubercular testing of the supernatants to identify those organisms in which antibiotic production is induced during competition with MTb. RNAseq of these organisms under inducing as well as non-inducing conditions to identify biosynthetic gene clusters in the genome that are induced by MTb coupled with whole genome sequencing is underway. In project 4, target identification for prioritized series is initiated by mutation frequency analysis, whole genome resequencing of resistant isolates, and metabolomics analyses. In addition, for top hits of interest where SAR indicates that certain positions on the molecule can be modified while retaining anti-tubercular activity, we have chemically modified the compounds by addition of a linker that can be UV-crosslinked onto the putative targets, as well as a linker moiety that provides a handle allowing purification of the resultant ligand-protein complexes. This chemical biology approach is guiding our efforts in target identification. Whole genome sequencing data combined with compound metabolite analyses have also highlighted the extensive repertoire of xenobiotic metabolizing enzymes that MTb possesses that either inactivate or in some cases activate the hit of interest. Our MTb metabolism screens of small molecule libraries that probe the diversity of compounds transformed by the pathogen and characterize the classes of enzymatic transformations has enabled us to identify unique enzymatic reactions that are not detectable in parallel screens of human microsome metabolism assays. An understanding of these metabolic processes will help us exploit or circumvent the activity of these enzymes in our drug development process. On a similar vein, we extending our studies to explore the role of cell wall and cell membrane associated proteins that facilitate the uptake of compounds including both nutrients and drugs. In project 5 we are continuing work to explore the importance of respiratory pathways, the biosynthesis of various cofactors, as well as MTb-specific metabolites such as mycocyclosin in maintaining viability under replicating, non-replicating and during pathogenesis of the host.